JPH0827181A - Purification of sialic acid-containing. clycoprotein - Google Patents
Purification of sialic acid-containing. clycoproteinInfo
- Publication number
- JPH0827181A JPH0827181A JP16644994A JP16644994A JPH0827181A JP H0827181 A JPH0827181 A JP H0827181A JP 16644994 A JP16644994 A JP 16644994A JP 16644994 A JP16644994 A JP 16644994A JP H0827181 A JPH0827181 A JP H0827181A
- Authority
- JP
- Japan
- Prior art keywords
- sialic acid
- glycoprotein
- mucin
- buffer solution
- porous beads
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、生理活性物質として知
られているシアル酸含有糖蛋白質を生体抽出物から工業
的に効率よく精製する方法に関する。TECHNICAL FIELD The present invention relates to a method for industrially efficiently purifying a sialic acid-containing glycoprotein known as a physiologically active substance from a biological extract.
【0002】[0002]
【従来の技術】シアル酸は、炭素9個を含むノイラミン
酸のアシル誘導体であるN−アセチルノイラミン酸、N
−グリコシルノイラミン酸、N,O−ジアセチルノイラ
ミン酸等及びこれらの誘導体の総称であり、生体内では
一般に糖蛋白質やガングリオシドのヘテロオリゴ糖の非
還元末端に結合して存在する。BACKGROUND OF THE INVENTION Sialic acid is N-acetylneuraminic acid, which is an acyl derivative of neuraminic acid containing 9 carbon atoms, N
-Glycosyl neuraminic acid, N, O-diacetylneuraminic acid and the like, and their derivatives, which are generally present in the living body bound to the non-reducing end of a glycoprotein or a ganglioside heterooligosaccharide.
【0003】シアル酸含有糖蛋白質に含まれるシアル酸
は、これを含有する糖蛋白質を安定化して、その生理活
性を維持し、且つ/又は種々の受容体(レセプター)と
の結合に重要な役割を果たしている。さらに、ムチン
は、その1分子中に多量のシアル酸が結合しており、胃
腸管や気管の内膜の機械的刺激に対する保護作用、消化
酵素に対する保護作用、細菌感染に対する防御作用、優
れた保湿作用及び乳化作用を有し、また、生体に対する
安全性も高いことが知られている。近年、これらのムチ
ンの特性に着目し、化粧品、医薬品(特に皮膚外用剤)
等の保湿成分として、さらには食品、化粧品、医薬品、
医薬部外品、トイレタリー等の各種産業分野において、
乳化剤としての利用の研究が進められている。Sialic acid contained in a sialic acid-containing glycoprotein stabilizes the glycoprotein containing the sialic acid, maintains its physiological activity, and / or plays an important role in binding to various receptors. Plays. In addition, mucin has a large amount of sialic acid bound in its molecule, and protects against mechanical irritation of the lining of the gastrointestinal tract and trachea, protects against digestive enzymes, protects against bacterial infections, and has excellent moisturizing effect. It is known that it has an action and an emulsifying action, and is highly safe for the living body. In recent years, attention has been paid to the characteristics of these mucins, and cosmetics and pharmaceuticals (especially external skin preparations)
As moisturizing ingredients such as, food, cosmetics, pharmaceuticals,
In various industrial fields such as quasi-drugs and toiletries,
Research on its use as an emulsifier is ongoing.
【0004】上記のような現状において、ムチン、その
他のシアル酸含有糖蛋白質を医薬、化粧品等に利用する
目的で、これらを工業的に大量に処理可能な精製法が望
まれている。Under the present circumstances as described above, for the purpose of utilizing mucin and other sialic acid-containing glycoproteins in medicines, cosmetics, etc., there is a demand for a purification method capable of industrially treating them in large quantities.
【0005】シアル酸含有糖蛋白質は、非常に粘度が高
く、特にシアル酸を多量に含有しているムチンは極めて
高粘度の物質である。シアル酸含有糖蛋白質に限らず、
粘性の高い物質を精製することは一般に非常に困難であ
る。それ故、かなり古くからシアル酸含有糖蛋白質を精
製しようとの試みが為されてきたが、未だ十分といえる
方法は見出されていないのが現状である。Sialic acid-containing glycoprotein has a very high viscosity, and mucin containing a large amount of sialic acid is an extremely high-viscosity substance. Not limited to sialic acid-containing glycoprotein,
Purification of highly viscous materials is generally very difficult. Therefore, although attempts have been made to purify sialic acid-containing glycoproteins for quite some time, the present situation is that no sufficient method has been found yet.
【0006】これまでに報告されているシアル酸含有糖
蛋白質の精製法としては、種々の生体成分、例えば血
液、尿、各種臓器を原料として、アルコール分画、アセ
トン分画、第4級アンモニウム塩、硫酸アンモニウム、
塩化ナトリウム等による分画を繰り返し、さらに種々の
陰イオン交換体、陽イオン交換体を用いたイオン交換ク
ロマトグラフィー、ハイドロキシアパタイトによる吸着
クロマトグラフィー、セファデックスG−100、セフ
ァデックスG−200、セファクリルS−300、セフ
ァロースCL4Bなどのゲル濾過クロマトグラフィーを
用いた方法又はこれらの組み合わせによる方法が挙げら
れる。[0006] The purification methods of sialic acid-containing glycoproteins reported so far include alcohol fractions, acetone fractions and quaternary ammonium salts using various biological components such as blood, urine and various organs as raw materials. , Ammonium sulfate,
Fractionation with sodium chloride and the like is repeated, and further ion exchange chromatography using various anion exchangers and cation exchangers, adsorption chromatography with hydroxyapatite, Sephadex G-100, Sephadex G-200, Sephacryl S. -300, a method using gel filtration chromatography such as Sepharose CL4B, or a method using a combination thereof.
【0007】なお、本願出願人は、限外濾過によるムチ
ンの精製方法を提案した(特開平5−310799
号)。この方法によれば、純度の高いムチンを一工程で
得ることができるが、この方法によってもシアル酸含有
糖蛋白質の純度の指標となるシアル酸/蛋白質(S/
P)比は1.03程度であり、また、大量処理は困難で
ある。The applicant of the present application has proposed a method for purifying mucin by ultrafiltration (JP-A-5-310799).
issue). According to this method, high-purity mucin can be obtained in one step, but this method can also be used as an index of the purity of the sialic acid-containing glycoprotein.
The P) ratio is about 1.03, and mass processing is difficult.
【0008】[0008]
【発明が解決しようとする課題】上記のように種々のシ
アル酸含有糖蛋白質の精製法が知られているが、いずれ
も操作に多くの手間と時間がかかり、精製の程度も不十
分であり、工業的に大量処理が可能であり、しかも安価
に精製が行える方法は未だ見出されていない。As described above, various purification methods for sialic acid-containing glycoproteins are known, but all of them require a lot of labor and time for operation, and the degree of purification is insufficient. However, a method capable of industrially performing large-scale processing and inexpensively purifying has not been found yet.
【0009】本発明は、上記現状に鑑み、シアル酸含有
糖蛋白質を短時間で、工業的に大量処理が可能であり、
且つ安価に精製できる方法を提供することを目的とす
る。In view of the above situation, the present invention is capable of industrially mass treating sialic acid-containing glycoproteins in a short time,
Moreover, it aims at providing the method which can be refine | purified at low cost.
【0010】[0010]
【課題を解決するための手段】本発明者らは、上記目的
を達成できるシアル酸含有糖蛋白質の精製法を開発すべ
く鋭意検討を重ねた結果、シアル酸がキトサンに特異的
に吸着されることを見出した。この現象に着目し、シア
ル酸含有糖蛋白質をキトサン多孔性ビーズに吸着させ、
精製を試みたところ、シアル酸含有糖蛋白質を短時間
で、工業的に大量に、安価に、しかも従来法に比べて高
純度に精製できることを見出し本発明を完成させた。Means for Solving the Problems The present inventors have conducted extensive studies to develop a method for purifying a sialic acid-containing glycoprotein that can achieve the above object, and as a result, sialic acid is specifically adsorbed by chitosan. I found that. Focusing on this phenomenon, the sialic acid-containing glycoprotein was adsorbed on the chitosan porous beads,
As a result of attempting purification, the inventors have found that sialic acid-containing glycoprotein can be purified in a short time, industrially in large quantities, at low cost, and with higher purity than conventional methods, and completed the present invention.
【0011】すなわち、本発明は、キトサン多孔性ビー
ズを用いることを特徴とするシアル酸含有糖蛋白質の精
製法を要旨とする。That is, the gist of the present invention is a method for purifying a sialic acid-containing glycoprotein characterized by using chitosan porous beads.
【0012】以下、本発明を詳細に説明する。まず、本
発明で用いるキトサン多孔性ビーズについて説明する。
キトサンとは、カニ、エビ、オキアミ及び昆虫類の甲皮
に含まれているN−アセチル−D−グルコサミンのβ−
1,4結合からなるキチンをN−脱アセチル化して得ら
れるポリグルコサミンである。キトサンの分子量は10
万以上であり、直鎖状の高分子物質で、多数のアミノ基
がその直鎖上に等間隔に存在し、天然物としては唯一の
カチオン性高分子として優れた特性を有するものであ
る。The present invention will be described in detail below. First, the chitosan porous beads used in the present invention will be described.
Chitosan is β-of N-acetyl-D-glucosamine contained in the crust of crab, shrimp, krill and insects.
It is a polyglucosamine obtained by N-deacetylating chitin composed of 1,4 bonds. Chitosan has a molecular weight of 10
It is a linear polymer substance having a large number of amino groups, and a large number of amino groups are present on the straight line at equal intervals, and has excellent properties as the only cationic polymer as a natural product.
【0013】キトサンの上記のような特性を利用して、
現在キトサンは種々の用途に応用されている。例えば、
固定化酵素用担体、アフィニティークロマトグラフィー
用担体、金属イオン吸着剤、細胞増殖用担体等の固体担
体として利用され、さらに、抗血液凝固剤、免疫強化
剤、外傷治療促進剤、人工臓器、抗菌剤、抗ウイルス剤
等の医療分野への応用も進められている。Utilizing the above characteristics of chitosan,
Currently, chitosan is used for various purposes. For example,
It is used as a solid carrier such as a carrier for immobilized enzyme, a carrier for affinity chromatography, a metal ion adsorbent, a carrier for cell proliferation, and further, an anticoagulant, an immunopotentiator, a wound healing accelerator, an artificial organ, an antibacterial agent. The application of antiviral agents to the medical field is also in progress.
【0014】近年、キトサンに種々の架橋処理を施すこ
とにより、酢酸、乳酸、ギ酸、クエン酸等の有機酸、塩
酸、硝酸等の無機酸及び水酸化ナトリウム等の塩基に不
溶性であり、種々の粒径、細孔径をもつビーズ状に成形
した多孔性ビーズが開発され、さらにキトサンの応用性
が広がりつつある。また、耐アルカリ性に優れ、水酸化
ナトリウムでの洗浄・再生が可能で、反復使用できるも
のも開発されている。By subjecting chitosan to various crosslinking treatments in recent years, it is insoluble in organic acids such as acetic acid, lactic acid, formic acid, and citric acid, inorganic acids such as hydrochloric acid and nitric acid, and bases such as sodium hydroxide. Porous beads formed into beads having a particle size and a pore size have been developed, and the applicability of chitosan is expanding. Also, it has been developed that has excellent alkali resistance, can be washed and regenerated with sodium hydroxide, and can be used repeatedly.
【0015】物質の精製にキトサン多孔性ビーズを用い
た応用例として、バイオ医薬品の製造過程で生じる不純
物である核酸やエンドトキシン(菌体内毒素)をキトサ
ン多孔性ビーズに吸着させて除去する方法が知られてい
るが、シアル酸含有糖蛋白質をキトサン多孔性ビーズに
吸着させて精製した例はない。As an application example of using chitosan porous beads for the purification of a substance, there is known a method of removing nucleic acids and endotoxin (endotoxin), which are impurities generated in the manufacturing process of biopharmaceuticals, by adsorbing them onto the chitosan porous beads. However, there is no example in which a sialic acid-containing glycoprotein is adsorbed on chitosan porous beads for purification.
【0016】本発明で精製されるべきシアル酸含有糖蛋
白質としては、例えば哺乳動物の顎下腺ムチン、胃粘膜
ムチン、卵白ムチン、免疫グロブリン類、フェツイン、
トランスフェリン、エリスロポエチン、サイログロブリ
ン、ガングリオシド又は赤血球膜糖蛋白質;哺乳動物の
脳下垂体前葉から得られる黄体形成ホルモン(LH)又
は卵胞刺激ホルモン(FSH)、尿中から得られる絨毛
性性腺刺激ホルモン(hCG)、閉経婦人尿性性腺刺激
ホルモン(hMG)、妊馬血清性性腺刺激ホルモン(P
MSG)等の性腺刺激ホルモンが挙げられる。The sialic acid-containing glycoprotein to be purified in the present invention includes, for example, mammalian submandibular gland mucin, gastric mucosa mucin, egg white mucin, immunoglobulins, fetuin,
Transferrin, erythropoietin, thyroglobulin, ganglioside or erythrocyte membrane glycoprotein; luteinizing hormone (LH) or follicle stimulating hormone (FSH) obtained from the anterior pituitary gland of mammals, chorionic gonadotropin (hCG) obtained from urine , Menopausal urinary gonadotropin (hMG), pregnant mare serum gonadotropin (P
Gonadotropins such as MSG).
【0017】本発明で用いることができるキトサン多孔
性ビーズとしては、pH4〜9の領域において水不溶性
に化学修飾されたキトサンからなるものが好ましい。精
製すべきシアル酸含有糖蛋白質の種類により、平衡化、
溶出に用いる緩衝液のpHを適宜選択すべきであるが、
通常上記範囲のpH領域において水不溶性であれば十分
である。The chitosan porous beads that can be used in the present invention are preferably those composed of chitosan chemically modified to be water-insoluble in the pH range of 4-9. Depending on the type of sialic acid-containing glycoprotein to be purified, equilibration,
The pH of the buffer used for elution should be selected appropriately,
Usually, it is sufficient if it is insoluble in water in the pH range of the above range.
【0018】キトサン多孔性ビーズの粒径、細孔径の平
均直径、かさ密度には特に制限はないが、それぞれ40
〜450μm、1〜10μm、30〜100mg/ml
程度が好ましい。The particle size of the chitosan porous beads, the average diameter of the pores, and the bulk density are not particularly limited, but they are 40 respectively.
~ 450 μm, 1-10 μm, 30-100 mg / ml
The degree is preferred.
【0019】上記のような特性を有し、本発明のシアル
酸含有糖蛋白質の精製に適した市販のキトサン多孔性ビ
ーズとしては、例えばクリムーバーII(栗田工業株式会
社製)、キトパールベーシック(富士紡績株式会社製)
等が挙げられる。Commercially available chitosan porous beads having the above-mentioned characteristics and suitable for purifying the sialic acid-containing glycoprotein of the present invention include, for example, CLIMOVER II (manufactured by Kurita Water Industries Ltd.) and Chitopearl Basic ( Fuji Spinning Co., Ltd.)
And the like.
【0020】次に、本発明のシアル酸含有糖蛋白質の精
製の操作手順について説明する。 (1)キトサン多孔性ビーズの調製 キトサン多孔性ビーズは、0.5〜2.0Mの水酸化ナ
トリウム溶液に懸濁し、一夜放置した後、蒸留水で洗浄
し、精製するシアル酸含有糖蛋白質の種類に応じてpH
4〜9の緩衝液で平衡化する。用いる緩衝液としては、
例えば酢酸緩衝液、クエン酸緩衝液、乳酸緩衝液、ギ酸
緩衝液、リン酸塩緩衝液、トリス塩酸緩衝液等が好まし
い。Next, an operational procedure for purifying the sialic acid-containing glycoprotein of the present invention will be described. (1) Preparation of Chitosan Porous Beads Chitosan porous beads were suspended in a 0.5 to 2.0 M sodium hydroxide solution, allowed to stand overnight, washed with distilled water, and purified to prepare a sialic acid-containing glycoprotein. PH depending on the type
Equilibrate with 4-9 buffers. The buffer used is
For example, acetic acid buffer, citrate buffer, lactate buffer, formate buffer, phosphate buffer, Tris-hydrochloric acid buffer and the like are preferable.
【0021】(2)粗シアル酸含有糖蛋白質の調製 本発明の方法により精製されるべき粗シアル酸含有糖蛋
白質は、生体成分から抽出し、不溶物を通常の濾過又は
遠心分離により除去したものでよい。さらに必要に応じ
てエタノール分画、アセトン分画、塩による分画を組み
込んで夾雑物を除去してもよい。(2) Preparation of Crude Sialic Acid-Containing Glycoprotein The crude sialic acid-containing glycoprotein to be purified by the method of the present invention is extracted from biological components and insoluble matter is removed by usual filtration or centrifugation. Good. Further, if necessary, an ethanol fraction, an acetone fraction, and a salt fraction may be incorporated to remove impurities.
【0022】(3)精製操作 上記(1)で平衡化したキトサン多孔性ビーズに粗シアル
酸含有糖蛋白質をキトサン多孔性ビーズの平衡化に用い
た緩衝液に溶解したものを添加し、シアル酸含有糖蛋白
質をキトサン多孔性ビーズに吸着させる。同緩衝液でキ
トサン多孔性ビーズを洗浄し、不純物を除去した後、シ
アル酸含有糖蛋白質の種類に応じて0.1〜0.5Mの
塩化ナトリウム、硫酸アンモニウム、硫酸ナトリウム、
塩化カリウム等によりイオン強度を高めた同緩衝液によ
りキトサン多孔性ビーズに吸着していたシアル酸含有糖
蛋白質を溶出させ、回収する。(3) Purification Procedure To the chitosan porous beads equilibrated in the above (1), the crude sialic acid-containing glycoprotein dissolved in the buffer used for equilibrating the chitosan porous beads was added, and sialic acid was added. The containing glycoprotein is adsorbed on the chitosan porous beads. After washing the chitosan porous beads with the same buffer to remove impurities, 0.1-0.5 M sodium chloride, ammonium sulfate, sodium sulfate, depending on the type of sialic acid-containing glycoprotein,
The sialic acid-containing glycoprotein adsorbed on the chitosan porous beads is eluted with the same buffer solution having an increased ionic strength with potassium chloride or the like and recovered.
【0023】ここで、精製の方式としては、キトサン多
孔性ビーズをカラムに充填するカラム方式、容器に平衡
化したキトサン多孔性ビーズを入れるバッチ方式のいず
れでも可能である。The purification method may be either a column method in which chitosan porous beads are packed in a column or a batch method in which equilibrated chitosan porous beads are placed in a container.
【0024】溶出したシアル酸含有糖蛋白質溶液は減圧
乾燥、噴霧乾燥、凍結乾燥等により乾燥され、目的の精
製シアル酸含有糖蛋白質末が得られる。The eluted sialic acid-containing glycoprotein solution is dried by vacuum drying, spray drying, freeze-drying or the like to obtain the desired purified sialic acid-containing glycoprotein powder.
【0025】さらに、上記溶出したシアル酸含有糖蛋白
質溶液を、ゲル濾過、電気透析、流水透析、限外濾過等
の公知の手段により脱塩処理した後、乾燥処理すること
により、塩を含まない精製シアル酸含有糖蛋白質末が得
られる。Further, the eluted sialic acid-containing glycoprotein solution is desalted by a known means such as gel filtration, electrodialysis, flowing water dialysis, ultrafiltration and the like, and then dried to obtain no salt. A purified sialic acid-containing glycoprotein powder is obtained.
【0026】[0026]
【実施例】以下、試験例及び実施例を挙げて本発明をさ
らに具体的に説明する。EXAMPLES The present invention will be described more specifically below with reference to test examples and examples.
【0027】試験例1:各種pHにおける種々のクロマ
トグラフィー担体のウシ顎下腺ムチンに対する吸着能の
検討 シアル酸含有糖蛋白質を効率よく精製するためには、ど
のようなクロマトグラフィー担体を用い、どのようなp
H下で精製を行うのが好ましいかを検討するため、以下
の担体及び緩衝液を用いてウシ顎下腺ムチンの精製を行
った。Test Example 1: Examination of Adsorption Ability of Various Chromatographic Carriers at Various pHs for Bovine Submandibular Mucin In order to efficiently purify sialic acid-containing glycoprotein, which chromatographic carrier was used, Like p
In order to investigate whether purification under H is preferable, bovine submandibular gland mucin was purified using the following carriers and buffers.
【0028】用いたクロマトグラフィー担体: DEAE−セファデックス CM−セファデックス(以上ファルマシア社製) クリムーバーII(栗田工業社製) ハイドロキシアパタイト(日本バイオラッド社製) 硫酸化セルロファイン(生化学工業社製) 用いた緩衝液:0.01M酢酸緩衝液(pH5.0及び
pH6.0) 0.01Mリン酸塩緩衝液(pH7.0) 0.01Mトリス塩酸緩衝液(pH8.0) 上記種々のpHの緩衝液で平衡化した上記各種のクロマ
トグラフィー担体をカラムに充填し、容量10mlのカ
ラムを作製した。Chromatographic carrier used: DEAE-Sephadex CM-Sephadex (all manufactured by Pharmacia) Crimover II (manufactured by Kurita Kogyo) Hydroxyapatite (manufactured by Nippon Bio-Rad) Sulfated Cellulofine (Seikagaku Corporation) Buffer used: 0.01 M acetate buffer (pH 5.0 and pH 6.0) 0.01 M phosphate buffer (pH 7.0) 0.01 M Tris-HCl buffer (pH 8.0) The above-mentioned various chromatographic carriers equilibrated with a pH buffer solution were packed in a column to prepare a column having a volume of 10 ml.
【0029】後記する実施例1の(1)に記載の粗ウシ顎
下腺ムチン(S/P比=1.03)100mgを種々の
緩衝液10mlに溶解した溶液をそれぞれのカラムに添
加した。次に、それぞれの非吸着画分と0.5M塩化ナ
トリウムを含む緩衝液で溶出される画分を集め、それぞ
れ透析により脱塩後、凍結乾燥末とした。得られたそれ
ぞれの画分の凍結乾燥末のシアル酸含量及び蛋白質含量
を測定し、シアル酸含量(S)/蛋白質含量(P)の比
(以下、S/P比という)を精製前の粗ムチンのS/P
比と比較した。結果を下記表1に示す。A solution prepared by dissolving 100 mg of crude bovine submandibular gland mucin (S / P ratio = 1.03) described in (1) of Example 1 described below in 10 ml of various buffers was added to each column. Next, each non-adsorbed fraction and the fraction eluted with a buffer containing 0.5 M sodium chloride were collected, desalted by dialysis, and then freeze-dried. The sialic acid content and protein content of the lyophilized powder of each of the obtained fractions were measured, and the ratio of sialic acid content (S) / protein content (P) (hereinafter referred to as S / P ratio) was determined before the purification. Mucin S / P
Compared to the ratio. The results are shown in Table 1 below.
【0030】[0030]
【表1】 注) ++:ムチンは全部担体に吸着され(すなわち非
吸着画分にはムチンは全く含まれず)、この吸着画分の
S/P比は原料の粗ムチンよりも上昇した。 +:ムチンは担体に吸着される画分と吸着されない画分
の両方に含まれ、S/P比は両画分とも原料の粗ムチン
と同等であった。 −:ムチンは担体に吸着されず(すなわち全てカラムを
素通りし)、この非吸着画分のS/P比は原料の粗ムチ
ンと同等であった。[Table 1] Note) ++: All the mucin was adsorbed on the carrier (that is, the non-adsorbed fraction did not contain mucin at all), and the S / P ratio of this adsorbed fraction was higher than that of the raw mucin. +: Mucin was contained in both the fraction adsorbed on the carrier and the fraction not adsorbed on the carrier, and the S / P ratio was equal to that of the crude mucin as the raw material in both fractions. -: Mucin was not adsorbed on the carrier (that is, all passed through the column), and the S / P ratio of this non-adsorbed fraction was equivalent to that of the raw mucin.
【0031】表1の結果から明らかなように、従来用い
られてきたクロマトグラフィー担体であるDEAE−セ
ファデックス等では、効率よくシアル酸含有糖蛋白質の
精製を行うことはできない。これに対し、pH5.0〜
6.0付近の緩衝液を用い、キトサン多孔性ビーズであ
るクリムーバーIIをクロマトグラフィー担体として用い
た場合には、他の担体を用いる場合に比べ、効率よくシ
アル酸含有糖蛋白質の精製が可能である。As is clear from the results shown in Table 1, it is not possible to efficiently purify the sialic acid-containing glycoprotein with the conventional chromatography carrier such as DEAE-Sephadex. On the other hand, pH 5.0-
When a buffer solution around 6.0 is used and the chitosan porous beads, CLIMOVER II, is used as the chromatography carrier, the sialic acid-containing glycoprotein can be purified more efficiently than when other carriers are used. Is.
【0032】特にpH5.0付近の緩衝液を用いた場合
に、原料の粗ムチンの全部が担体に吸着され、それを
0.5Mの塩化ナトリウムを含む緩衝液で溶出した画分
のS/P比が原料の粗ムチンよりも上昇しており、ムチ
ンを含むシアル酸含有糖蛋白質の精製に特に適してい
る。Especially when a buffer solution having a pH of about 5.0 was used, all of the crude mucin as a raw material was adsorbed on the carrier, and the S / P fraction of the fraction eluted with the buffer solution containing 0.5 M sodium chloride was used. The ratio is higher than that of raw mucin, which is a raw material, and is particularly suitable for purification of sialic acid-containing glycoprotein containing mucin.
【0033】試験例2:各種pHにおけるキトサン多孔
性ビーズカラムのウシ顎下腺ムチンに対する吸着能の検
討 上記試験例1で、キトサン多孔性ビーズがシアル酸含有
糖蛋白質の精製に特に適したクロマトグラフィー担体で
あることが明らかとなった。本試験例2では、キトサン
多孔性ビーズでシアル酸含有糖蛋白質を精製するための
好ましい条件を検討した。クフォマトグラフィー担体と
してキトサン多孔性ビーズであるクリムーバーII(栗田
工業社製)を用い、緩衝液として0.01M酢酸緩衝液
を用い、緩衝液のpHを下記表2のように変化させた。Test Example 2: Examination of Adsorption Ability of Chitosan Porous Beads Column to Bovine Submandibular Mucin at Various pHs In the above Test Example 1, the chitosan porous beads were particularly suitable for purification of glycoproteins containing sialic acid. It became clear that it was a carrier. In this test example 2, preferable conditions for purifying a sialic acid-containing glycoprotein with chitosan porous beads were examined. The chitosan porous beads Climover II (produced by Kurita Kogyo Co., Ltd.) was used as the chromatograph carrier, and 0.01 M acetate buffer was used as the buffer, and the pH of the buffer was changed as shown in Table 2 below. .
【0034】各種pHの緩衝液で平衡化した担体をカラ
ムに充填し、容量10mlのカラムを作製した。後記す
る実施例1の(1)の粗ウシ顎下腺ムチン(S/P比=
1.03)500mgを各種pHの緩衝液10mlに溶
解させた溶液をそれぞれのカラムに添加した。次にそれ
ぞれの非吸着画分、0.25M塩化ナトリウムを含む緩
衝液で溶出される画分及び0.5M塩化ナトリウムを含
む緩衝液で溶出される画分を集め、透析により脱塩後、
凍結乾燥末とした。得られたそれぞれの画分の収率及び
S/P比を下記表2に示した。The carrier equilibrated with buffer solutions of various pHs was packed in a column to prepare a column having a volume of 10 ml. The crude bovine submandibular gland mucin of (1) of Example 1 described later (S / P ratio =
1.03) A solution of 500 mg dissolved in 10 ml of a buffer solution having various pH was added to each column. Next, the respective non-adsorbed fractions, the fraction eluted with the buffer containing 0.25M sodium chloride and the fraction eluted with the buffer containing 0.5M sodium chloride were collected, and after desalting by dialysis,
It was freeze-dried. The yield and S / P ratio of each obtained fraction are shown in Table 2 below.
【0035】[0035]
【表2】 [Table 2]
【0036】表2の結果から、pH6の緩衝液を用いた
非吸着画分、及びpH4.0〜6.0の緩衝液を用いた
0.25M塩化ナトリウム含有緩衝液で溶出する画分に
シアル酸含有糖蛋白質が多く含まれ、収率(回収率)及
び純度(S/P比)の点からpH4〜9付近の0.1〜
0.5M塩化ナトリウムに相当するイオン強度を有する
緩衝液好ましいと考えられる。特にpH5.5の緩衝液
を用い、0.25M塩化ナトリウムに相当するイオン強
度を有する緩衝液で溶出するのが好ましいことがわか
る。表2中、特に好ましい条件で精製を行った場合、ム
チンの収率は64%と非常に高く、S/P比も1.06
以上と高い。From the results shown in Table 2, it was found that the non-adsorbed fraction using the pH 6 buffer solution and the fraction eluted with the 0.25 M sodium chloride-containing buffer solution using the pH 4.0 to 6.0 buffer solution were sialylated. A large amount of acid-containing glycoprotein is contained, and from the viewpoint of yield (recovery rate) and purity (S / P ratio), a pH value of around pH 4-9 is 0.1-0.1.
A buffer having an ionic strength corresponding to 0.5 M sodium chloride is considered preferable. It can be seen that it is particularly preferable to use a buffer having a pH of 5.5 and elute with a buffer having an ionic strength corresponding to 0.25M sodium chloride. In Table 2, when purified under particularly preferable conditions, the yield of mucin was 64%, which was very high, and the S / P ratio was 1.06.
Above and high.
【0037】以上の試験例1及び2の結果を踏まえ、好
ましい本発明のキトサン多孔性ビーズを用いたシアル酸
含有糖蛋白質の精製法を具体的に説明する。Based on the results of the above Test Examples 1 and 2, the preferred method for purifying a sialic acid-containing glycoprotein using the chitosan porous beads of the present invention will be specifically described.
【0038】実施例1:ウシ顎下腺ムチンの精製 (1)粗ウシ顎下腺ムチン 特開平5−310799号公報の実施例1に記載の限外
濾過により精製して得られたウシ顎下腺ムチンを原料
(以下、「粗ムチン」という。)として用いた。この粗
ムチンの3%水溶液におけるシアル酸(S)含量は0.
69%、蛋白質(P)含量は0.67%(乾燥末に換算
すると、シアル酸(S)含量は23%、蛋白質(P)含
量は22.3%となる。)であり、S/P比は1.03
であった。Example 1: Purification of bovine submandibular gland mucin (1) Crude bovine submandibular gland mucin Purified bovine submandibular gland obtained by ultrafiltration as described in Example 1 of JP-A-5-310799 Gland mucin was used as a raw material (hereinafter referred to as "crude mucin"). The sialic acid (S) content in a 3% aqueous solution of this crude mucin was 0.
69%, protein (P) content 0.67% (converted to dry powder, sialic acid (S) content 23%, protein (P) content 22.3%), S / P Ratio is 1.03
Met.
【0039】(2)キトサン多孔性ビーズによる精製 キトサン多孔性ビーズ(栗田工業株式会社製、クリムー
バーII)を2M水酸化ナトリウム溶液に懸濁して一夜放
置したものを、カラム(口径120mm、長さ12c
m)に充填し、450mlのキトサン多孔性ビーズを充
填したカラムを作製した。カラム中のキトサン多孔性ビ
ーズをpH5.5の0.01M酢酸緩衝液で平衡化し
た。(2) Purification with Chitosan Porous Beads Chitosan porous beads (Kurimova Co., Ltd., CLIMOVER II) were suspended in a 2M sodium hydroxide solution and allowed to stand overnight. The column (diameter 120 mm, length) 12c
m) was filled with 450 ml of chitosan porous beads. The chitosan porous beads in the column were equilibrated with 0.01 M acetate buffer pH 5.5.
【0040】キトサン多孔性ビーズカラムに、粗ムチン
末23gをpH5.5の0.01M酢酸緩衝液2リット
ルに溶解した溶液を添加した。さらに、粗ムチン溶液を
ポンプでカラム内に循環させてムチンをキトサン多孔性
ビーズに吸着させた。その後、pH5.5の0.01M
酢酸緩衝液10リットルをポンプでカラム内に循環させ
て不純物をキトサン多孔性ビーズから洗い出した。To a chitosan porous bead column, a solution prepared by dissolving 23 g of crude mucin powder in 2 liters of 0.01 M acetate buffer having a pH of 5.5 was added. Further, the crude mucin solution was circulated in the column by a pump to adsorb mucin to the chitosan porous beads. Then 0.01M at pH 5.5
Impurities were washed out from the chitosan porous beads by pumping 10 liters of acetate buffer through the column.
【0041】次いで、キトサン多孔性ビーズに吸着した
ムチンを0.25M塩化ナトリウムを含むpH5.5の
0.01M酢酸緩衝液10リットルで溶出した。Next, the mucin adsorbed on the chitosan porous beads was eluted with 10 liters of a 0.01 M acetate buffer having a pH of 5.5 containing 0.25 M sodium chloride.
【0042】ムチンを含む溶出液を限外濾過装置(アミ
コン社製、ダイアフローホローファイバーシステムDC
4型、カートリッジH1P100使用)を用いて濃縮・
脱塩した後、凍結乾燥して精製ムチン末15g(収率:
65.2%)を得た。The eluate containing mucin is ultrafiltered (Amicon, Diaflow hollow fiber system DC
4 type, using cartridge H1P100)
After desalting, it was freeze-dried and purified mucin powder 15 g (yield:
65.2%) was obtained.
【0043】得られた精製ムチン末のシアル酸(S)含
量は32.0%、蛋白質(P)含量は27.0%であ
り、S/P比は1.19であった。また、分子量分布
は、10万以上が99.0%、10万以下が1.0%で
あった。シアル酸含量はエールリッヒ法、蛋白質含量は
ローリー法、分子量分布はTSK gel G3000
SWカラム(東ソー社製)をもちいた高速液体クロマト
グラフィー法によって測定した。The purified mucin powder obtained had a sialic acid (S) content of 32.0%, a protein (P) content of 27.0%, and an S / P ratio of 1.19. Further, the molecular weight distribution was 99.0% for 100,000 or more and 1.0% for 100,000 or less. Sialic acid content is Ehrlich method, protein content is Lowry method, molecular weight distribution is TSK gel G3000
It was measured by a high performance liquid chromatography method using a SW column (manufactured by Tosoh Corporation).
【0044】現在までに報告されている精製ムチンのS
/P比の最高値は(実験室レベルの精製により得られた
ものであるが)1.20付近である。上記本発明の精製
法によれば、工業的規模での精製で、S/P比=1.1
9という高い純度のムチンが得られた。しかも、実験室
レベルの精製では、種々の操作を繰り返し、長時間を要
する。これに対し、本発明の精製法によれば、極短時間
で、簡易な操作で、大量に高純度のムチンを得ることが
できる。S of purified mucin reported so far
The highest value of the / P ratio (although obtained by laboratory-level purification) is around 1.20. According to the above-described purification method of the present invention, S / P ratio = 1.1 in purification on an industrial scale.
Mucin having a high purity of 9 was obtained. Moreover, in the laboratory-level purification, various operations are repeated and it takes a long time. On the other hand, according to the purification method of the present invention, a large amount of high-purity mucin can be obtained by a simple operation in an extremely short time.
【0045】実施例2:脳下垂体性性腺刺激ホルモンの
精製 (1)粗脳下垂体前葉抽出物末の調製 脳下垂体前葉(250g)を細切したものを、pH8.
5の30%アセトン溶液で抽出し、濾過により不溶物を
除去した抽出液に塩酸を加えてpH4.5に調整し、生
じた沈殿を濾過により除去した。濾液にアセトンを加え
てアセトン濃度70%とし、目的物を含むアセトン沈殿
を形成させた。得られたアセトン沈殿を、脱水後、減圧
乾燥し、粗脳下垂体前葉抽出物末(1g)を得た。Example 2: Purification of pituitary gonadotropin (1) Preparation of crude anterior pituitary extract powder Finely chopped anterior pituitary (250 g) had a pH of 8.
The extract was extracted with a 30% acetone solution of 5 and the insoluble matter was removed by filtration, hydrochloric acid was added to adjust the pH to 4.5, and the generated precipitate was removed by filtration. Acetone was added to the filtrate to an acetone concentration of 70% to form an acetone precipitate containing the target substance. The obtained acetone precipitate was dehydrated and then dried under reduced pressure to obtain a crude anterior pituitary gland extract powder (1 g).
【0046】(2)キトサン多孔性ビーズによる精製 キトサン多孔性ビーズは、実施例1の(2)と同様に処理
してpH5.5の0.01M酢酸緩衝液で平衡化したも
のをカラムに充填し、10mlのカラムを作製した。(2) Purification with chitosan porous beads Chitosan porous beads were treated in the same manner as in (2) of Example 1 and equilibrated with 0.01 M acetate buffer of pH 5.5, and packed in a column. Then, a 10 ml column was prepared.
【0047】粗脳下垂体前葉抽出物末100mgをpH
5.5のの0.01M酢酸緩衝液10mlに溶解した溶
液をキトサン多孔性ビーズカラムに添加した後、pH
5.5の0.01M酢酸緩衝液50mlでカラムを洗浄
し、0.25M塩化ナトリウムを含む同緩衝液100m
lで溶出する画分を集めた。得られた画分を透析により
脱塩した後、凍結乾燥し、精製脳下垂体性性腺刺激ホル
モン末を得た。100 mg of crude anterior pituitary extract powder was adjusted to pH.
After adding a solution of 5.5 of 0.01 M acetate buffer in 10 ml to a chitosan porous bead column,
The column was washed with 50 ml of 0.01M acetate buffer of 5.5, and 100m of the same buffer containing 0.25M sodium chloride.
Fractions eluting at 1 were collected. The obtained fraction was desalted by dialysis and then lyophilized to obtain a purified pituitary gonadotropin powder.
【0048】また、フリードマン試験により、得られた
精製脳下垂体性性腺刺激ホルモン末の比活性を測定し
た。精製末の収量、フリードマン試験による比活性及び
活性回収率を下記表3に示した。Further, the specific activity of the obtained purified pituitary gonadotropin powder was measured by the Friedman test. The yield after purification, the specific activity by the Friedman test and the activity recovery rate are shown in Table 3 below.
【0049】比較例:DEAE−イオン交換体及びCM
−イオン交換体による脳下垂体性性腺刺激ホルモンの精
製 キトサン多孔性ビーズカラムの代わりにDEAE−イオ
ン交換体(DEAE−セルロファイン、生化学工業社
製)カラム及びCM−イオン交換体(CM−セファデッ
クス、ファルマシア社製)カラムを用い、集める精製脳
下垂体性性腺刺激ホルモンを含む画分又は溶出溶液等を
下記のように変えた以外は上記実施例2と同様に処理を
行ない、それぞれ精製脳下垂体性性腺刺激ホルモン末を
得た。Comparative Example: DEAE-ion exchanger and CM
-Purification of pituitary gonadotropin by ion exchanger Instead of chitosan porous bead column, DEAE-ion exchanger (DEAE-Cellulofine, Seikagaku Corporation) column and CM-ion exchanger (CM-Sepha). (Dex, Pharmacia) column was treated in the same manner as in Example 2 except that the fraction containing the purified pituitary gonadotropin or the elution solution was changed as follows, and each purified brain was used. The pituitary gonadotropin powder was obtained.
【0050】DEAE−イオン交換体カラムにおいて
は、目的物を含む画分が粗脳下垂体前葉抽出物溶液10
mlをカラムに添加したときの非吸着画分であるため、
この非吸着画分を集めた。CM−イオン交換体カラムに
おいては、粗脳下垂体前葉抽出物溶液10mlをカラム
に添加し、0.1M塩化ナトリウムを含むpH5.5の
0.01M酢酸緩衝液50mlでカラムを洗浄した後、
0.5M塩化ナトリウムを含む同緩衝液100mlで溶
出する画分を集めた。In the DEAE-ion exchanger column, the fraction containing the target substance was a crude anterior pituitary extract solution 10
Since it is the non-adsorbed fraction when ml is added to the column,
This non-adsorbed fraction was collected. In the CM-ion exchanger column, 10 ml of the crude anterior pituitary extract solution was added to the column, and the column was washed with 50 ml of 0.01 M acetate buffer of pH 5.5 containing 0.1 M sodium chloride.
Fractions eluted with 100 ml of the same buffer containing 0.5 M sodium chloride were collected.
【0051】得られた精製脳下垂体性性腺刺激ホルモン
末の収量、フリードマン試験による比活性及び活性回収
率を下記表3に示す。The yield of the purified pituitary gonadotropin powder obtained, the specific activity by the Friedman test and the activity recovery rate are shown in Table 3 below.
【0052】[0052]
【表3】 注)原料の粗脳下垂体前葉抽出物末の比活性は、2単位
/mgである。[Table 3] Note) The specific activity of the raw powder of crude anterior pituitary gland extract is 2 units / mg.
【0053】上記表3の結果から、他のクロマトグラフ
ィー担体を用いるよりも、キトサン多孔性ビーズを用い
ることにより、脳下垂体性性腺刺激ホルモンを効率的
に、且つ高度に精製することができることは明らかであ
る。From the results in Table 3 above, it can be seen that pituitary gonadotropin can be efficiently and highly purified by using chitosan porous beads rather than by using other chromatographic carriers. it is obvious.
【0054】[0054]
【発明の効果】本発明のキトサン多孔性ビーズを用いる
ことを特徴とするシアル酸含有糖蛋白質の精製法によれ
ば、シアル酸含有糖蛋白質を短時間で、工業的に大量
に、安価に、しかも従来法に比べて高純度に精製するこ
とが可能になった。According to the method for purifying a sialic acid-containing glycoprotein characterized by using the chitosan porous beads of the present invention, the sialic acid-containing glycoprotein can be produced in a short time, industrially in large quantities, and at low cost. Moreover, it has become possible to purify to a higher purity than the conventional method.
Claims (9)
徴とするシアル酸含有糖蛋白質の精製法。1. A method for purifying a sialic acid-containing glycoprotein, which comprises using chitosan porous beads.
下腺ムチン、胃粘膜ムチン、卵白ムチン、免疫グロブリ
ン類、フェツイン、トランスフェリン、エリスロポエチ
ン、サイログロブリン、ガングリオシド及び赤血球膜糖
蛋白質からなる群から選ばれる請求項1に記載の精製
法。2. The sialic acid-containing glycoprotein is selected from the group consisting of mammalian submandibular gland mucin, gastric mucosa mucin, egg white mucin, immunoglobulins, fetuin, transferrin, erythropoietin, thyroglobulin, ganglioside and erythrocyte membrane glycoprotein. The purification method according to claim 1, wherein
チンである請求項2に記載の精製法。3. The method according to claim 2, wherein the sialic acid-containing glycoprotein is bovine submandibular gland mucin.
下垂体前葉から得られる黄体形成ホルモン(LH)又は
卵胞刺激ホルモン(FSH)、尿中から得られる絨毛性
性腺刺激ホルモン(hCG)、閉経婦人尿性性腺刺激ホ
ルモン(hMG)及び妊馬血清性性腺刺激ホルモン(P
MSG)からなる群より選ばれる性腺刺激ホルモンであ
る請求項1に記載の精製法。4. A sialic acid-containing glycoprotein, wherein luteinizing hormone (LH) or follicle stimulating hormone (FSH) obtained from the anterior pituitary gland of a mammal, chorionic gonadotropin (hCG) obtained from urine, Menopausal urinary gonadotropin (hMG) and pregnant mare serum gonadotropin (P
The purification method according to claim 1, which is a gonadotropin selected from the group consisting of MSG).
腺刺激ホルモンである請求項4に記載の精製法。5. The method according to claim 4, wherein the sialic acid-containing glycoprotein is pituitary gonadotropin.
〜9の領域において水不溶性に化学修飾されたキトサン
からなる請求項1に記載の精製法。6. The chitosan porous beads used have a pH of 4
The purification method according to claim 1, which comprises chitosan chemically modified to be water-insoluble in the region of -9.
酸含有糖蛋白質を0.1〜0.5Mの塩化ナトリウム、
硫酸アンモニウム、硫酸ナトリウム又は塩化カリウムを
含む緩衝液により溶出する請求項1に記載の精製法。7. A sialic acid-containing glycoprotein adsorbed on chitosan porous beads, wherein the sialic acid-containing glycoprotein is 0.1-0.5 M sodium chloride,
The purification method according to claim 1, which is eluted with a buffer solution containing ammonium sulfate, sodium sulfate, or potassium chloride.
液、乳酸緩衝液、ギ酸緩衝液、リン酸塩緩衝液及びトリ
ス塩酸緩衝液からなる群より選ばれる請求項7に記載の
精製法。8. The purification method according to claim 7, wherein the buffer solution is selected from the group consisting of acetate buffer solution, citrate buffer solution, lactate buffer solution, formate buffer solution, phosphate buffer solution and Tris-HCl buffer solution. .
る請求項1に記載の精製法。9. The purification method according to claim 1, wherein the purification method is a column method or a batch method.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP16644994A JPH0827181A (en) | 1994-07-19 | 1994-07-19 | Purification of sialic acid-containing. clycoprotein |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP16644994A JPH0827181A (en) | 1994-07-19 | 1994-07-19 | Purification of sialic acid-containing. clycoprotein |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0827181A true JPH0827181A (en) | 1996-01-30 |
Family
ID=15831620
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP16644994A Pending JPH0827181A (en) | 1994-07-19 | 1994-07-19 | Purification of sialic acid-containing. clycoprotein |
Country Status (1)
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JP (1) | JPH0827181A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2012050175A1 (en) | 2010-10-15 | 2012-04-19 | 日本ケミカルリサーチ株式会社 | Method for producing glycoprotein having mannose residue as non-reducing end of sugar chain |
KR101485551B1 (en) * | 2012-12-11 | 2015-01-22 | 서울대학교산학협력단 | Separation of ovotransferrin from chicken egg white without using solvents |
KR20170010862A (en) | 2014-05-31 | 2017-02-01 | 니홍 케미칼 리써치 가부시키가이샤 | Culture medium containing uridine and n-acetyl-d-mannosamine |
CN111035581A (en) * | 2019-12-31 | 2020-04-21 | 嘉必优生物技术(武汉)股份有限公司 | Method for maintaining stability of sialic acid solution at high temperature and application thereof |
-
1994
- 1994-07-19 JP JP16644994A patent/JPH0827181A/en active Pending
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2012050175A1 (en) | 2010-10-15 | 2012-04-19 | 日本ケミカルリサーチ株式会社 | Method for producing glycoprotein having mannose residue as non-reducing end of sugar chain |
KR20130119932A (en) | 2010-10-15 | 2013-11-01 | 니홍 케미칼 리써치 가부시키가이샤 | Method for producing glycoprotein having mannose residue as non-reducing end of sugar chain |
US11649474B2 (en) | 2010-10-15 | 2023-05-16 | Jcr Pharmaceuticals Co., Ltd. | Method for producing glycoprotein having mannose residue as non-reducing end of sugar chain |
KR101485551B1 (en) * | 2012-12-11 | 2015-01-22 | 서울대학교산학협력단 | Separation of ovotransferrin from chicken egg white without using solvents |
KR20170010862A (en) | 2014-05-31 | 2017-02-01 | 니홍 케미칼 리써치 가부시키가이샤 | Culture medium containing uridine and n-acetyl-d-mannosamine |
US10273448B2 (en) | 2014-05-31 | 2019-04-30 | Jcr Pharmaceuticals Co., Ltd. | Medium containing uridine and N-acetyl-D-mannosamine |
US10557115B2 (en) | 2014-05-31 | 2020-02-11 | Jcr Pharmaceuticals Co., Ltd. | Medium containing uridine and N-acetyl-D-mannosamine |
CN111035581A (en) * | 2019-12-31 | 2020-04-21 | 嘉必优生物技术(武汉)股份有限公司 | Method for maintaining stability of sialic acid solution at high temperature and application thereof |
CN111035581B (en) * | 2019-12-31 | 2023-04-14 | 嘉必优生物技术(武汉)股份有限公司 | Method for maintaining stability of sialic acid solution at high temperature and application thereof |
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